Journal of Experimental Botany, Vol. 54, No. 383, pp. 801±812, February 2003
DOI: 10.1093/jxb/erg084
RESEARCH PAPER
Mapping of QTL associated with nitrogen storage and
remobilization in barley (Hordeum vulgare L.) leaves
Suzanne Mickelson1, Deven See2, Fletcher D. Meyer1, John P. Garner1, Curt R. Foster1, Tom K. Blake3 and
Andreas M. Fischer1,4
1
Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717-3150, USA
2
Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA
International Center for Agricultural Research in the Dry Areas, PO Box 5466, Aleppo, Syrian Arab Republic
3
Received 19 June 2002; Accepted 22 October 2002
Abstract
Nitrogen uptake and metabolism are central for vegetative and reproductive plant growth. This is re¯ected
by the fact that nitrogen can be remobilized and
reused within a plant, and this process is crucial for
yield in most annual crops. A population of 146
recombinant inbred barley lines (F8 and F9 plants,
grown in 2000 and 2001), derived from a cross
between two varieties differing markedly in grain
protein concentration, was used to compare the location of QTL associated with nitrogen uptake, storage
and remobilization in ¯ag leaves relative to QTL controlling developmental parameters and grain protein
accumulation. Overlaps of support intervals for such
QTL were found on several chromosomes, with
chromosomes 3 and 6 being especially important.
For QTL on these chromosomes, alleles associated
with inef®cient N remobilization were associated with
depressed yield and higher levels of total or soluble
organic nitrogen during grain ®lling and vice versa;
therefore, genes directly involved in N recycling or
genes regulating N recycling may be located on
these chromosomes. Interestingly, the most prominent QTL for grain protein concentration (on chromosome 6) did not co-localize with QTL for nitrogen
remobilization. However, QTL peaks for nitrate and
soluble organic nitrogen were detected at this locus
for plants grown in 2001 (but not in 2000). For these,
alleles associated with low grain protein concentration were associated with higher soluble nitrogen
levels in leaves during grain ®lling; therefore, gene(s)
found at this locus might in¯uence the nitrogen sink
strength of developing barley grains.
4
Key words: Barley, grain protein concentration, Hordeum
vulgare L., nitrogen remobilization, nitrogen storage, QTL,
yield.
Introduction
Nitrogen is quantitatively the most important mineral
nutrient taken up from the soil by plants (Marschner,
1995). A thorough understanding of its assimilation,
transport and metabolism is central to modern plant
biology, both with respect to understanding basic plant
function and with respect to breeding or engineering crop
plants for desirable traits.
The most important source of nitrogen for many crops
(with the remarkable exception of legumes capable of
symbiotic nitrogen ®xation) is nitrate. Depending on the
plant species and its physiological status, nitrate can be
stored or reduced and assimilated in both roots and leaves
(Marschner, 1995). In cereals, nitrate assimilation is
predominantly localized in the shoots (Cooper and
Clarkson, 1989; Larsson et al., 1991). In leaf vacuoles,
nitrate can accumulate to high levels, which is undesirable
if vegetative plant parts are used for food or animal feed
(Marschner, 1995). The largest fraction of assimilated
nitrogen is used for protein synthesis, while smaller
fractions are present in other macromolecules (nucleic
acids) and in a large number of different primary and
secondary metabolites (Peoples and Dalling, 1988).
Among these, amino acids, and especially the amides
(glutamine, asparagine), occupy a central position, both in
terms of the role they play in metabolism (Ireland and Lea,
1999) and because of their importance for long-distance
To whom correspondence should be addressed. Fax: +1 406 994 7600. E-mail: ®[email protected]
Journal of Experimental Botany, Vol. 54, No. 383, ã Society for Experimental Biology 2003; all rights reserved
802 Mickelson et al.
transport of reduced nitrogen in the phloem (Bush, 1999).
In mesophyll cells of fully developed cereal leaves, over
50% of the total nitrogen is present in the photosynthetic
apparatus in chloroplasts (Peoples and Dalling, 1988). In
many annual crops including cereals, nitrogen recycled
from senescing vegetative plant parts during grain-®lling is
important for nitrogen yield (Feller and Fischer, 1994).
Because of its importance for crop physiology, senescence
has been intensively studied using biochemical and
molecular approaches (Feller and Fischer, 1994; Quirino
et al., 2000), but a comprehensive understanding of the
regulation of this complex developmental process has not
yet been achieved.
Barley (Hordeum vulgare L.), besides its importance as
a crop, is an established model plant for both genetic and
physiological studies (Koornneef et al., 1997; Forster et al.,
2000). Some aspects of plant nitrogen metabolism have
been studied in detail in this species (Warner and
Kleinhofs, 1992; Sueyoshi et al., 1995; Botrel and
Kaiser, 1997; Kronzucker et al., 1999; Vidmar et al.,
2000), and well-de®ned genetic maps are available
(Forster et al., 2000). The focus of this study was the
analysis of causal relationships between leaf nitrogen
metabolism and grain protein accumulation, using a
combination of quantitative genetic and biochemical
tools. A population of 146 recombinant inbred lines
(RILs), derived from a cross between two varieties with
marked, highly heritable differences in grain protein
concentration, was used for this investigation. A 110
point linkage map has previously been established for
these lines and used to map QTL controlling grain protein
concentration (See et al., 2002). Here, total nitrogen was
assayed in ¯ag leaves at three developmental stages from
anthesis through maturity to monitor total nitrogen accumulation and remobilization. These data were complemented with the analysis of nitrates and amino nitrogen
(amino acids, small peptides) to follow continued uptake
of inorganic nitrogen into the leaves as well as levels of
transportable N compounds after anthesis. QTL and
correlative analyses on these parameters were used and
compared to QTL obtained for grain protein concentration
to identify loci important for cereal nitrogen recycling.
Materials and methods
Plant material
Two barley (Hordeum vulgare L.) varieties, `Lewis' (CI15856) and
`Karl'(CI15487), were chosen as parents for this study based on
marked differences in grain protein concentration at maturity. Karl is
a six-rowed variety which produces grain of consistently lower
protein concentration than most other varieties. Lewis is a
commonly grown two-rowed barley. A 146-member recombinant
inbred population was developed by single seed descent from a cross
and used to map the genes responsible for barley grain protein
concentration (See et al., 2002). For the present study, F8 and F9
plants were grown in two independent replicates in a randomized
block design near Bozeman, MT in summer 2000 and 2001 using
standard farming practice. Flag leaves (10 leaves per sample) from
all 146 lines as well as the parental lines, Lewis and Karl, were
collected around anthesis, mid-grain ®ll and plant maturity, immediately frozen in liquid nitrogen, transported to the laboratory in
liquid nitrogen and stored at ±80 °C. Leaves were weighed, ground
to a ®ne powder in liquid nitrogen using a mortar and pestle, and
stored again at ±80 °C until analysis.
The fact that heading date is one of the parameters which
segregated in this population (see Results) made it impossible to
collect samples for all lines at exactly the same developmental
stages. Therefore, the dates were chosen in such a way that the ®rst
sampling time point was at anthesis 63 d for most lines, before any
potential changes of leaf nitrogen metabolism associated with the
main phase of grain-®lling. The last harvest date was characterized
by mature plants, and leaves were fully senesced for all lines. Some
in¯uence of the developmental stage on the assayed parameters is to
be expected for the middle harvest date.
Total nitrogen quanti®cation
Total leaf nitrogen was analysed using a combustion method in a
Leco FP 528 protein analyser (Leco Corporation, St Joseph, MI).
Approximately 100 mg leaf powder was used, the exact weight
determined using an analytical balance, and the sample was
processed according to the manufacturer's instructions. The determined nitrogen concentrations (percentage of total sample weight)
were used for correlative and QTL analysis (see below).
Additionally, as leaf weights change during development and
senescence, measured leaf weights were used to calculate total
nitrogen on a per-leaf basis (mg leaf±1) and to determine changes in
total leaf nitrogen between anthesis and maturity (DN, mg leaf±1).
Quanti®cation of nitrates and soluble a-NH2 nitrogen
For the extraction of soluble nitrogenous compounds, 50±100 mg of
leaf powder was heated at 80 °C for 15 min in 500 ml (<75 mg) or
1000 ml H2O (>75 mg) in a 1.5 ml tube, extracted with a small pestle
(®tting the conical bottom of the micro tube) using a motor unit for
30 s, again heated to 80 °C for 15 min, centrifuged at full speed in an
Eppendorf centrifuge, and supernatants were used for nitrate and
amino nitrogen quanti®cation, either directly or after storage at
±80 °C.
ÿ
For nitrate analysis, NOÿ
3 was reduced to NO2 enzymatically,
using immunoaf®nity-puri®ed corn leaf nitrate reductase (The
Nitrate Elimination Co. [NECi], Lake Linden, MI) as outlined by
the manufacturer's protocol, and NOÿ
2 was determined colorimetrically using the sulphanilamide±N-naphthylethylenediamine
method (NECi, Lake Linden, MI). Optical densities were determined
after 10 min at 540 nm in a SPECTRAmax PLUS384 spectrophotometer (Molecular Devices, Sunnyvale, CA). The method was
calibrated using 0±10 nmol KNO3.
A solution of 150 ppm TNBS (2,4,6-trinitrobenzene sulphonic
acid) in 50 mM Na-borate buffer pH 9.5 (150 ml per well; total assay
volume including sample: 200 ml) was used to assay soluble a-NH2
nitrogen. The method was calibrated using 0±50 nmol glycine.
Optical densities were measured at 405 nm 60 min after the addition
of TNBS reagent.
Statistical and QTL analysis
Data were collected for both independent replications for all
nitrogen uptake, storage, and remobilization traits from both years
(2000 and 2001) and analysed using the General Linear Model
procedure of SAS (SAS Institute Inc., 1990). Phenotypic correlations
were calculated among traits using least square mean values for each
genotype combined across replications and environments. Narrow
QTL analysis of nitrogen remobilization 803
sense heritability on an entry-mean basis for the recombinant inbred
lines was determined for these traits using the following equation:
oÂA2 /(oÂ2G+oÂ2GE/E+oÂ2e/RE)
where oÂA2 represents additive genetic variance, oÂ2G represents
genotypic variance, oÂ2GE/E represents genotype by environmental
variance divided by the number of environments, and oÂ2e/RE
represents error variance divided by the number of replications
multiplied by the number of environments.
The 110 point linkage map developed by See et al. (2002) for the
population of barley lines used in this study was used for genetic
analyses. This map is based mostly on AFLP markers anchored to
linkage maps with previously mapped morphological, storage
protein and SNP markers (Kleinhofs et al., 1993; Liu et al., 1996;
Kuenzel et al., 2000). QTL analysis was conducted using the
PlabQTL Version 1.1 mapping program (Utz and Melchinger,
1996). Composite interval mapping employing the covariate
SELECT option of PlabQTL was performed for detection of QTL.
This option uses step-wise multiple regression to select cofactors.
For QTL model building and detection of QTL3environment
interactions, a LoD threshold of 2.5 was used. The additive effect of
a marker was calculated by PlabQTL as ((mean of the homozygous
Karl class±mean of the homozygous Lewis class)/2). QTL support
intervals are calculated as the point along the signi®cance peak at
which the LoD score is 1.0 unit less than the peak LoD score. The
phenotypic variance (s2p) explained by a single QTL was estimated
by the square of the partial correlation coef®cient (R2). The
phenotypic variance (s2p) explained by the QTL model was estimated
by the adjusted correlation coef®cient (R2adj), which accounts for the
number of predictors in the QTL model.
QTL analyses of grain yield, heading date and grain protein
concentrations have been published previously for this population
(See et al., 2002). These data have been adapted and re-analysed
using composite interval mapping to demonstrate relationships
between leaf and grain nitrogen composition, plant development and
metabolism.
Results
Genotypic variation and heritability
A wide range of values was observed in the recombinant
inbred lines (RILs) for all traits measured in this study
(Figs 1, 2), although differences between the parental lines
Lewis and Karl, which were originally selected for their
highly heritable difference in grain protein concentration,
were smaller for most parameters, demonstrating transgressive segregation in both directions.
Speci®cally, both mean values and distribution for total
¯ag leaf nitrogen were comparable for the ®rst (around
anthesis) and second (mid-grain ®ll) harvests, with a mean
value of ~1.6% total leaf nitrogen (based on fresh weight)
at anthesis and 1.45% at mid-grain ®ll (Fig. 1A, B). By
contrast, much higher variation, from 1.25 to >3%, was
found at maturity (Fig. 1C). As the leaves were fully
senesced and partially desiccated at this point, this re¯ects
a lower average per-leaf nitrogen content than on the
previous dates (data not shown). The high variation among
lines for nitrogen remaining in leaves at maturity re¯ects
differences in nitrogen remobilization (which may be
partially explained by differences in structural N content),
suggesting that this population can be used for the genetic
analysis of this parameter.
Figure 1D shows differences (mg leaf±1) in total leaf
nitrogen content between the ®rst (anthesis) and last (plant
maturity) harvest dates. Although leaf nitrogen metabolism is a dynamic process, characterized by both import and
export of nitrogen compounds, this parameter gives an
indication of the quantity of nitrogen exported from ¯ag
leaves to developing grains. Again, a wide range of
variation, from <1 to 2.5 mg leaf±1, was observed, with a
mean of ~1.5 mg leaf±1.
Soluble inorganic (nitrate; Fig. 1E, F) and organic
(amino nitrogen; Fig. 1G, H) nitrogen compounds were
quanti®ed to gain a better understanding of overall
nitrogen metabolism in the leaves. Freshly acquired
nitrogen is primarily imported through the xylem in the
form of nitrate in cereal leaves (Marschner, 1995), whereas
amino acids are the main transport forms used to export
nitrogen from both mature and senescing leaves (Bush,
1999). Nitrates varied from ~5 to 65 ppm (based on fresh
weight) around anthesis (Fig. 1E) and continued to
accumulate in leaves during grain ®ll, reaching concentrations of 300 ppm in some lines by mid-grain ®ll
(Fig. 1F). As for the other nitrogen-related parameters,
considerable variation in leaf nitrate was obvious in the
population used for this study. The distribution for nitrate
at anthesis appears slightly skewed on the linear scale
used; this skewness can be eliminated and the data appear
normally distributed when a logarithmic scale is used (not
shown). Similar concentrations and distributions of amino
nitrogen were found at anthesis and mid-grain ®ll (Fig. 1G,
H), but overall concentrations and means were slightly
lower at the later harvest date.
Leaf nitrogen parameters were compared with a number
of developmental (leaf weight, heading date, yield)
parameters and with grain protein concentration (Fig. 2A±
D). Leaf fresh weights at mid-grain ®ll (Fig. 2A) were
considered representative of leaf size, as they correlated
well with leaf weights at anthesis (not shown).
Correlations between leaf weights at mid-grain ®ll and
maturity were less signi®cant (not shown), as different
parameters (e.g. remobilization ef®ciency) become more
important at the later time point. Leaf size, grain protein
percentage, grain yield, and ¯owering date all varied
widely among lines in this population. Leaf weights ranged
from 80 to >200 mg leaf±1 (Fig. 2A), while heading dates
ranged from 2 July to 12 July (between 182 and 192 Julian
days, Fig. 2B), and grain protein concentration ranged
from 11% to 17% (Fig. 2C). Yield ranged from 1.5 t ha±1 to
5.5 t ha±1 (Fig. 2D).
Narrow-sense heritability was calculated for leaf nitrogen parameters and leaf weight. For leaf nitrogen concentration (% of FW) it was found to be 34% at anthesis, 57%
at mid-grain ®ll and 48% at maturity. For difference
in leaf nitrogen content between anthesis and maturity
804 Mickelson et al.
Fig. 1. Frequency distribution of leaf nitrogen parameters in the 146 recombinant inbred barley lines used for this study. Mean values for the
recombinant inbred lines (RILs, open triangle) as well as the parental lines, `Lewis' and `Karl' (closed triangles) are shown.
(mg leaf ±1), it was found to be 49%, for nitrate (ppm) at
anthesis 19%, but only 13% at mid-grain ®ll. The value for
soluble a-NH2 nitrogen was 38% at anthesis and 34% at
mid-grain ®ll. Narrow-sense heritability was 65% for leaf
weight.
An analysis of variance was performed on all leaf
nitrogen parameters. This analysis indicated that suf®cient variation exists between RILs to detect genetic
mechanisms controlling the traits (data not shown). Only
leaf nitrate at mid-grain ®ll did not exhibit signi®cant (at
P <0.05) genotypic variation, a result of a larger range
of values in 2000 relative to 2001. Signi®cant (P <0.01)
genotype by environment interactions were detected for
nitrogen at maturity, difference in leaf nitrogen concentration between anthesis and maturity, leaf nitrate at
mid-grain ®ll and leaf weight. Phenotypic and rank
correlations between years were highly signi®cant and
positive for the traits with genotype by environment
QTL analysis of nitrogen remobilization 805
Fig. 2. Frequency distribution of developmental parameters and grain protein concentration in the 146 recombinant inbred barley lines used for
this study. Mean values for the recombinant inbred lines (RILs, open triangle) as well as the parental lines, `Lewis' and `Karl' (closed triangles)
are shown.
interactions. No crossover interactions were observed
and interactions were mainly associated with differences
in magnitude of effects between years. Genotypic
variance exceeded genotype by environmental variance
for all traits with the exception of leaf nitrate at midgrain ®ll. As the G3E interactions detected were
predominantly caused by differences in magnitude
between years, data were combined across environments
for further analysis.
Correlation analysis
The focus of this work was the evaluation of the genetic
control of nitrogen storage and remobilization in barley
leaves, as related to grain protein concentration and protein
yield. To achieve this goal, simple statistical correlations
(Table 1) were determined in addition to genetic analysis
in the population of 146 RILs.
Interestingly, nitrogen remobilization ef®ciency from
¯ag leaves (measured both by residual N in leaves at plant
maturity and by the difference in leaf nitrogen content
between anthesis and maturity, DN) was not correlated
with grain protein concentration (Table 1). On the other
hand, nitrogen remobilization ef®ciency was positively
correlated with total yield and protein yield (calculated
from protein concentration and yield) suggesting that
remobilized nitrogen positively in¯uences yield, but does
not control grain protein concentration. Poor ¯ag leaf
nitrogen remobilization became apparent at a relatively
early time point, since total leaf nitrogen as well as soluble
inorganic (nitrate) and organic nitrogen pools were higher
at mid-grain ®lling in those lines retaining high nitrogen
concentrations in leaves at maturity. Total leaf nitrogen at
anthesis was positively correlated with soluble a-NH2
nitrogen at the same time point and total leaf nitrogen at
maturity, but negatively correlated with nitrate at midgrain ®ll.
As noticed by other researchers (Forster et al., 2000; See
et al., 2002), developmental parameters have a strong
in¯uence on physiological traits. Heading date was
negatively correlated with nitrogen remobilization from
leaves and yield, but positively correlated with grain
protein concentration. Nitrogen was more ef®ciently
retranslocated in barley lines with large ¯ag leaves;
accordingly, leaf weight was also positively correlated
with yield.
Overall, the picture emerging from simple correlative
analysis of the 146 RILs used in this study indicates that,
while nitrogen remobilization from leaves is important for
total yield and protein yield, grain protein concentration is
not controlled by this parameter. The correlations found
between heading date and nitrogen pool sizes in leaves
indicate that nitrogen remobilization is more complete in
those lines with a larger time span between ¯owering and
plant maturity.
806 Mickelson et al.
Table 1. Trait correlations
Mean values of two years were used to correlate leaf nitrogen (% of FW) at anthesis, mid-grain ®ll and maturity, difference in leaf nitrogen
content (DN, mg leaf±1) between anthesis and maturity, leaf nitrate (ppm) at anthesis and mid-grain ®ll, soluble a-NH2 nitrogen (mmol g±1 FW)
in leaves at anthesis and mid-grain ®ll, grain protein concentration (%), yield (t ha±1), heading date (Julian days), leaf weight (mg leaf±1) and
protein yield (kg ha±1).
Trait
Nitrogen at
anthesis
Nitrogen at
mid-grain ®ll
Nitrogen at
maturity
DN
NO3± at anthesis
NO3± at
mid-grain ®ll
a-NH2 at
anthesis
a-NH2 at
mid-grain ®ll
Leaf weight
Heading date
Grain protein
Yield
Protein yield
Nitrogen Nitrogen Nitrogen DN
at
at mid- at maturity
anthesis grain ®ll
Heading Grain
NO3± at NO3± at a-NH2 at a-NH2 at Leaf
anthesis midanthesis mid-grain weight date
protein
grain ®ll
®ll
Yield
Protein
yield
0.148
0.220** 0.609**
±0.036 ±0.454** ±0.640**
0.042 ±0.196* 0.039
±0.213** 0.385** 0.422**
0.373** 0.148
0.135
±0.382**
±0.145
±0.039
0.160
0.154
0.072
0.690** 0.429**
±0.332**
0.457**
±0.018
±0.419**
±0.443**
±0.480**
0.291**
±0.035
±0.393**
±0.417**
0.234**
±0.275** 0.390**
±0.045
0.035
±0.311** ±0.047
0.650**
±0.287**
±0.070
0.231**
0.212*
0.110
±0.365**
±0.185*
0.054
±0.022
0.014
0.397** 0.139
±0.115
0.221**
±0.061
±0.344**
±0.377**
±0.106
0.129
0.069
±0.110
±0.086
±0.295**
0.324** ±0.069
±0.057
±0.002 0.465**
±0.387** 0.170* ±0.484** ±0.279**
±0.433** 0.036 ±0.293** 0.181* 0.890**
*, ** Signi®cant at P <0.05 and P <0.01, respectively.
QTL analysis
Physiological and morphological traits may be positively
or negatively correlated for several reasons. Species like
barley that are predominantly self-pollinated contain
varieties that differ for a plethora of traits, and mere
comparison of inbred lines fails to uncover which of these
correlations are the result of pleiotropy or linkage, and
which are the result of divergent gene ®xation in different
lineages. Distinguishing between these two alternative
explanations of trait correlation is important because
pleiotropy and tight linkage make selection for correlation-breaking individuals dif®cult. If an allele resulting in
low foliar nitrate content pleiotropically results in low
grain or forage yield, then that allele will be eliminated in
crop improvement programmes. If, however, one genotype
contains alleles at two unlinked genes that independently
contribute to poor forage yield and low foliar nitrate
content, then selection of desirable high-yielding, low
foliar nitrate recombinants becomes straightforward.
The purpose of this study was the analysis of relationships between genes controlling variation in leaf nitrogen
metabolism and grain protein accumulation, using a
combination of quantitative genetic and biochemical
tools. Since data from the two years of experimentation
were pooled, QTL 3 environment interactions were
analysed. Such interactions were detected for nitrogen at
mid-grain ®ll, nitrogen at maturity, and leaf nitrate at midgrain ®ll. In all cases, the interaction was associated with
decreased effects of the QTL in 2001 attributed to
decreased variance for the traits in 2001 relative to 2000.
Based on total ¯ag leaf nitrogen levels at plant maturity
and/or differences in total leaf nitrogen between anthesis
and maturity (DN), several statistically signi®cant QTL for
nitrogen remobilization were detected on the seven barley
chromosomes (Table 2; Fig. 3), with chromosomes 3, 6
and 7 each contributing at least two loci. The regions
around markers acat466 and acag135 on chromosome 3,
acag273 on chromosome 4, TB2122 on chromosome 5,
and acgc132 and acgt517 on chromosome 6 appear to be of
special interest, since the support intervals of QTL relevant
for residual leaf N or nitrogen remobilization overlap with
those of other QTL relevant for nitrogen metabolism
(Fig. 3), namely with total or partial (nitrate, soluble aNH2) leaf nitrogen pools at earlier assay dates. For the loci
on chromosomes 3 and 6, alleles associated with low
residual N at leaf maturity were also associated with
depressed total or soluble organic N levels at earlier
harvest dates, while high residual N correlated with higher
N levels at earlier dates. This relationship was inverted at
acag273 on chromosome 4, where those lines carrying the
`Karl' allele, demonstrating higher residual leaf N at
maturity, had depressed soluble a-NH2 nitrogen levels at
mid-grain ®ll, and at TB2122 on chromosome 5, where
lines with lower residual leaf N at maturity had enhanced
leaf N at anthesis (Table 2; Fig. 3). In the latter region, an
interesting additional observation is that both total leaf
QTL analysis of nitrogen remobilization 807
Table 2. Quantitative trait loci (QTL) for leaf nitrogen parameters
Total leaf nitrogen (% of FW) at anthesis, mid-grain ®ll and maturity, difference in leaf nitrogen content (DN, mg leaf±1) between anthesis and
maturity, nitrate (ppm) at anthesis and mid-grain ®ll, and a-NH2 nitrogen (mmol g±1 FW) in leaves at anthesis and mid-grain ®ll are shown.
Analyses were based on mean values from two years. Allelic effect shows the effect of carrying the `Karl' as opposed to the `Lewis' allele in
the respective position, using the units indicated.
Trait
Number of
QTLs
Chromosome
number
Nearest
marker
Support
interval
(cM)
LoD
Explained
variance
(%)
Nitrogen at anthesis
2
Nitrogen at mid-grain ®ll
6
Nitrogen at maturity
8
DN
3
NO3± at anthesis
6
NO3± at mid-grain ®ll
a-NH2 at anthesis
a-NH2 at mid-grain ®ll
1
1
8
5
6
3
3
5
6
6
7
3
3
4
5
6
6
7
7
6
7
7
1
3
3
4
6
7
6
2
3
3
3
3
4
6
6
7
actc410
acgc132
acat466
acag135
acgc424
actt298
acgc132
actc55
acat466
acag135
acag273
TB2122
acgg515
acgt517
acaa389
rachi
acgc132
acgc140
pinb1
actg256
acaa158
acag135
HVM40
actt298
acaa270
actt298
VVLOCI
acat466
actg385
actc135
acag175
acag273
actt298
acgt517
acaa327
124±184
66±90
182±208
298±318
54±70
0±12
70±102
24±28
176±208
260±308
206±238
172±194
54±76
140±152
42±56
142±190
68±90
32±46
52±64
282±306
14±59
252±322
0±36
0±20
212±244
0±16
140±154
178±210
306±332
348±366
496±512
220±238
0±14
134±160
240±244
2.97
4.40
3.44
6.77
3.08
5.67
3.40
3.32
3.52
4.99
2.93
4.45
2.83
2.97
3.13
2.66
4.70
4.26
3.59
3.03
3.23
2.58
3.70
2.78
3.49
5.51
4.69
2.66
4.73
3.29
3.00
4.14
3.45
3.01
5.53
8.9
13.0
10.3
19.2
9.3
16.6
10.3
10.0
10.5
14.6
8.9
13.1
8.6
8.9
9.5
8.1
13.9
12.7
10.8
9.2
9.7
7.8
12.1
8.5
10.6
16.2
13.8
8.1
13.9
9.8
9.1
12.3
10.4
9.1
16.3
nitrogen at anthesis and yield are enhanced in presence of
`Karl' alleles. Additional QTL associated with N remobilization from ¯ag leaves were found around markers
acaa389 and Rachi on chromosome 7, but these were not
associated with other N metabolism-relevant QTL.
It is well-known from the literature that most of the
nitrogen found in proteins of mature cereal grains is
remobilized and retranslocated from senescing vegetative
plant parts (Peoples and Dalling, 1988; Feller and Fischer,
1994). In this study, correlation analysis, while demonstrating a positive correlation between nitrogen remobilization and total yield as well as protein yield, did not link
N remobilization (DN) from leaves with grain protein
concentration (expressed as % of grain weight), suggesting
that grain protein concentration is not controlled by the
amount of nitrogen remobilized from the leaves.
Accordingly, no overlaps of support intervals for these
traits were found by QTL analysis. On the other hand,
Total
explained
variance
(%)
11.9
43.3
29.1
20.0
34.9
16.2
13.8
37.7
Allelic
effect
0.046
±0.037
0.031
0.049
0.033
0.040
±0.036
0.122
0.113
0.197
0.130
±0.126
±0.120
±0.147
±0.169
0.177
0.142
0.151
0.148
3.321
±3.950
±3.616
±3.819
3.906
4.219
34.549
±2.761
1.170
1.740
1.258
±1.160
±1.381
1.333
±1.464
1.627
while there was also no overlap of support intervals for DN
and yield, support intervals of QTL for yield and leaf
nitrogen at maturity (in % of FW) overlap on chromosomes 3 (near acag155/acat466) and 5 (actc410/TB2122).
On chromosome 3, alleles associated with high leaf N at
maturity are associated with low yield, while on chromosome 5, alleles associated with low residual leaf N are also
associated with high yield.
Besides QTL associated with leaf nitrogen remobilization, a few additional loci interesting for nitrogen metabolism were identi®ed. Several chromosomal regions
involved in nitrate accumulation were found on chromosomes 1 (around marker actg256), 3 (acaa158 and
acag135), 4 (HVM40), 6 (actt298) and 7 (acaa270). This
information may prove useful for further investigations
into nitrogen acquisition, long-distance and cellular transport, and for breeding low-nitrate forage barley varieties.
From a basic point of view, the area at actt298 on
808 Mickelson et al.
Fig. 3. Linkage map of RILs showing the location of QTL associated with the contents of total leaf nitrogen (% of fresh weight) at anthesis, midgrain ®ll and maturity; differences in total leaf nitrogen content (DN, mg leaf±1) between anthesis and maturity; leaf nitrate (ppm) at anthesis and
mid-grain ®ll; soluble a-NH2 nitrogen (mmol g FW±1) at anthesis and mid-grain ®ll; leaf weight (mg leaf±1) at mid-grain ®ll; heading date (Julian
days); grain protein concentration (%) and yield (t ha±1). Length of bar corresponds to support interval for each QTL, determined as outlined in
`Materials and methods'. The in¯uence of the presence of the `Karl' allele (+ or ±) on the observed trait is indicated below the support interval of
each QTL.
chromosome 6 is especially interesting, as several QTL
for leaf nitrate at anthesis and mid-grain ®ll, soluble
a-NH2 and total leaf nitrogen at mid-grain ®ll overlap,
suggesting that this region of chromosome 6 may
contain gene(s) involved in leaf nitrogen acquisition and
accumulation.
QTL analysis of nitrogen remobilization 809
Table 3. Quantitative trait loci (QTL) for leaf weight (mg leaf±1), heading date (Julian days), grain protein concentration (%) and
yield (t ha±1)
Analyses were based on mean values from two years. Allelic effect shows the effect of carrying the `Karl' as opposed to the `Lewis' allele in
the respective position, using the units indicated.
Trait
Number of
QTLs
Chromosome
number
Nearest
marker
Support
interval
(cM)
LoD
Explained
variance
(%)
Leaf weight
2
Heading date
4
Grain protein
2
Yield
8
1
6
1
2
2
6
2
6
1
2
2
3
3
5
6
7
actt285
acgc132
actg600
acaa267
acaa210
acgt517
hvbkasi
HVM74
HVM5
acaa210
VVLOCI
acag155
acgc469
actc410
actt166
acaa327
262±278
72±96
16±52
8±26
60±68
132±148
0±18
250±256
216±254
48±70
128±146
166±186
324±340
142±174
214±226
238±244
4.62
4.54
3.36
7.42
2.57
5.75
8.66
19.50
3.59
4.13
3.66
4.56
7.18
4.05
4.85
4.40
13.6
13.4
10.1
23.0
5.8
16.6
26.3
45.9
10.8
12.3
11.0
13.4
20.3
12.0
14.2
13.2
Correlative analysis (Table 1) pointed to a positive
correlation between heading date and grain protein concentration, and to negative correlations between heading
date and nitrogen remobilization (DN) and yield. This is
re¯ected by con®dence interval overlaps of QTL for
heading date and grain protein on chromosome 2 (at
hvbkasi), for heading date and yield also on chromosome 2
(at acaa210), and for heading date and leaf N at maturity
near acgt517 on chromosome 6 (Fig. 3). For all these loci,
the observed allelic effects (Tables 2, 3) con®rm the results
of correlative analysis, and demonstrate the in¯uence of
plant development on physiological traits.
The region around the major grain protein QTL at
HVM74 on chromosome 6 (Table 3; Fig. 3; compare See
et al., 2002) was analysed in more detail. While no
additional QTL above the threshold of 2.5 were detected in
this chromosomal region using mean values of two years
of experimentation (2000 and 2001), detailed separate
analysis of each year (data not shown) allowed some
interesting observations. The 2001 ®eld data showed a
QTL peak with a LoD of 5.51 and a support interval from
246±258 cM for nitrates at mid-grain ®ll, and a QTL peak
with a LoD of 3.68 and a support interval from 246±254
cM for soluble a-NH2 nitrogen. No signi®cant peak was
found for nitrates at mid-grain ®ll analysing the 2000 data;
while a peak for soluble a-NH2 nitrogen was found close
to HVM74 in this dataset (LoD=3.4, support interval from
266±278 cM), it did not overlap with the support interval
of the grain protein QTL. Interestingly, for the 2001 data,
`Karl' alleles associated with low grain protein concentration at maturity were associated with higher nitrate and
soluble a-NH2 nitrogen at mid-grain ®ll, and RILs
Total
explained
variance (%)
17.7
35.5
50.2
38.1
Allelic
effect
±10.546
8.899
0.495
±1.050
0.621
±1.012
±0.606
±0.727
±0.147
±0.227
0.275
±0.201
±0.249
0.170
0.176
±0.162
carrying the `Karl' allele in the adjacent QTL for soluble
a-NH2 nitrogen in 2000 also showed depressed values.
While these QTL peaks are not stable under changing
environmental conditions, it appears intriguing that, for
2001, they co-localize with the grain protein concentration
peak explaining 40% of the variation in this trait, and their
interpretation may prove helpful in understanding the
regulation of cereal grain protein concentration.
Discussion
The focus of this paper was to map QTL important for
nitrogen storage and recycling from senescing leaves to the
grains after anthesis. These data, especially if combined
with QTL data for grain protein yield and grain protein
concentration, can be used for ongoing breeding efforts
aimed at in¯uencing grain protein concentration while
maintaining maximal yield. In the past, several studies
have pointed to a negative correlation between these two
parameters in cereals (Beninati and Busch, 1992; and
references cited therein). Typically, a low grain protein
concentration is advantageous if barley is used for malting,
while high grain protein is a trait usually sought if this crop
is used for food or animal feed (Weston et al., 1993).
Additionally, the approach chosen here represents a ®rst
step towards the identi®cation of genes important for
nitrogen redistribution from senescing vegetative tissues.
Gene identi®cation should be facilitated by recently
published results from rice genome sequencing and
synteny between the different cereal genomes (Smilde
et al., 2001; Goff et al., 2002).
810 Mickelson et al.
Nitrogen remobilization from barley ¯ag leaves
Several QTL in¯uencing nitrogen remobilization (DN)
and/or nitrogen concentration in leaves at maturity were
identi®ed on chromosomes 3, 4, 5, 6 and 7 (Table 2; Fig. 3).
Con®dence intervals for some of these QTL overlapped
with QTL for other nitrogen metabolism-related parameters and for developmental parameters (heading date, leaf
size).
Chromosomes 3 and 6 appear to be of special interest in
this context. Both contain regions where support intervals
of QTL for total leaf N at maturity overlap with QTL for
leaf nitrogen at earlier analysis dates. Interestingly, alleles
associated with low N in fully senesced leaves at these loci
are also associated with low soluble organic or total leaf N
at anthesis or mid-grain ®ll and vice versa; therefore, genes
either directly involved in nitrogen remobilization or genes
regulating this process may be present in those chromosomal regions. A draft sequence of the rice genome has
recently been published and used to study synteny among
the cereal genomes (Goff et al., 2002; and supplementary
data to this article published online regarding synteny
between rice, corn and other cereals including barley). The
most interesting ®nding of these authors for the present
study is the fact that a high degree of synteny exists
between barley chromosome 3 and rice chromosome 1;
this result con®rms an earlier publication by Smilde et al.
(2001). Some aspects of the genetics of leaf nitrogen
accumulation and recycling have been studied by other
authors in rice (Obara et al., 2001; Ishimaru et al., 2001;
Yamaya et al., 2002). QTL determining the content of
Rubisco, soluble proteins and NADH-GOGAT as well as a
structural gene for NADH-GOGAT have been identi®ed
on rice chromosome 1 (Ishimaru et al., 2001; Obara et al.,
2001). Considering the synteny between rice chromosome
1 and barley chromosome 3, it appears possible that some
of these rice QTL are due to the same gene(s) or group of
genes as the QTL identi®ed in this study. Additional work
will be needed to identify the regions of the rice genome
syntenous to the parts of barley chromosome 6 discussed
here, as the data of Goff et al. (2002) indicate that regions
of a number of rice chromosomes contain markers derived
from barley chromosome 6. So far, a combination of
biochemical and molecular approaches has led to the
identi®cation of a large number of genes involved in
nitrogen recycling (Buchanan-Wollaston, 1997; Quirino
et al., 2000), but for many of them, their exact cellular
function remains elusive. It appears realistic that a
combination of biochemical and genetic approaches, as
used here, will contribute to the improvement of this
situation.
In addition to the information gained from N metabolism-related QTL overlaps, the in¯uence of developmental
parameters (heading date, leaf size) on nitrogen recycling
appears intriguing. Correlative analysis indicates a positive
correlation between heading date and leaf N at maturity
(i.e. late-¯owering lines are less ef®cient at N remobilization), and a negative correlation between leaf N concentration at maturity and leaf size (i.e. lines with large leaves
are more ef®cient at N remobilization). Regions with
support interval overlaps for these traits were found on
chromosomes 6 (acgc132, acgt517) and 7 (acaa389).
Pleiotropic effects of developmental genes on physiological parameters have been described in the literature
(Forster et al., 2000), and See et al. (2002; compare Fig. 3)
observed the in¯uence of heading date on grain protein
concentration. These observations could be explained if
it is assumed that the velocity of nitrogen export from
vegetative barley tissue does not segregate in the barley
population used, therefore making the time available for
nitrogen recycling more important. In this context, it is
interesting that Dreccer et al. (1997) found that they
were unable to affect the rate of N concentration
decline in wheat stems and leaves using a variety of
different treatments; based on these results, they
discussed the possibility of an `intrinsic' rate of nitrogen
export.
Leaf nitrogen metabolism and yield parameters
The in¯uence of nitrogen remobilization from senescing
vegetative plant parts on grain protein concentration and
yield has been investigated by different research groups
(Van Sanford and MacKown, 1987; Papakosta and
Gagianas, 1991; Beninati and Busch, 1992; Youngquist
and Maranville, 1992; Barneix and Guitman, 1993;
Oscarson et al., 1995; Lohaus et al., 1998; and references
cited therein). While the speci®c results depend on the
species/cultivar and the experimental system (®eld versus
controlled conditions) chosen, it appears reasonable to
conclude that leaf nitrogen metabolism in¯uences grain
protein concentration and/or grain protein yield.
In this study, no correlation between nitrogen remobilization ef®ciency and grain protein concentration was
detected (Table 1). On the other hand, a strong positive
correlation was found between N remobilization and total
yield as well as protein yield. As 50±90% of the nitrogen
found in cereal grains at harvest are derived from nitrogen
remobilized from vegetative plant parts (Van Sanford and
MacKown, 1987; Peoples and Dalling, 1988; Papakosta
and Gagianas, 1991) and photosynthetic proteins such as
Rubisco are usually degraded early during leaf senescence
(Feller and Fischer, 1994), leading to a decrease in
photosynthetic rates, this result may appear contradictory.
However, there is good experimental evidence that leaf
lipids, especially from plastidial membranes, are remobilized as well during leaf senescence (Gut and Matile, 1988;
McLaughlin and Smith, 1995); therefore, the results
obtained here may be explained if the lines more ef®cient
in N remobilization are ef®cient at lipid degradation and C
retranslocation as well. Considerable variation in the
QTL analysis of nitrogen remobilization 811
contribution of pre-anthesis assimilates to grain yield has
been found among different wheat varieties (Papakosta
and Gagianas, 1991), and Gallagher et al. (1975, 1976)
concluded that pre-anthesis storage of carbohydrates was
important for grain yields in barley and wheat.
As discussed for nitrogen remobilization, both grain
protein concentration and yield are in¯uenced by heading
date. This is re¯ected by a negative correlation between
heading date and yield, a positive correlation between
heading date and grain protein concentration (Fig. 3) and
by the co-localization of QTL for these traits. One of the
major QTL for grain protein concentration overlaps with a
QTL in¯uencing heading date (on chromosome 2), and the
support interval for a heading date QTL overlaps with a
yield QTL near marker acaa210, also on chromosome 2.
Therefore, it appears that in late-¯owering lines, there is a
more severe reduction in carbohydrate (starch) than in
protein yield, leading to fewer or smaller grains, but with a
higher protein concentration.
The single most important QTL for grain protein
concentration on chromosome 6 (at marker HVM74)
does not co-localize with any other signi®cant QTL, as
determined from two years of experimentation. However,
the fact that signi®cant QTL peaks, overlapping with the
support interval of this QTL for grain protein concentration, were found for both leaf nitrates and soluble organic
nitrogen at mid-grain ®ll in the 2001 dataset appears
intriguing. For both nitrates and soluble a-NH2, leaf levels
at mid-grain ®ll were higher in those lines carrying the
`Karl' allele, leading to lower grain protein concentration
at maturity (data not shown). It is tempting to speculate
that developing grains of lines carrying the `Lewis' allele
might be stronger N sinks; however, as narrow-sense
heritability of soluble leaf nitrogen pools (especially
nitrate) at mid-grain ®ll is lower than some of the other
measured parameters, this effect may be dif®cult to
observe. Since the recently characterized grain protein
QTL from Triticum turgidum (Joppa et al., 1997; Chee
et al., 2001) represents a potential homologue (See et al.,
2002), further genetic analysis of this chromosomal region
appears important, both from a basic (mechanistic understanding of grain protein accumulation) and applied
(importance of grain protein concentration as a quality
factor) point of view.
Acknowledgements
The authors would like to thank the Cereal Quality laboratory of
Montana State University for the use of their Leco FP-528 Protein
Analyser. This manuscript has been assigned Journal Series No.
2002-41, Montana Agricultural Experiment Station, Montana State
University-Bozeman. This work has been partially supported by
award number 0101019 from the USDA-NRI Competitive Grants
Program to AMF.
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